Polymeric gene delivery

ABSTRACT

A means for obtaining efficient introduction of exogenous genes into a patient, with long term expression of the gene, is disclosed. The gene, under control of an appropriate promoter for expression in a particular cell type, is encapsulated or dispersed with a biocompatible, preferably biodegradable polymeric matrix, where the gene is able to diffuse out of the matrix over an extended period of time, for example, a period of three to twelve months or longer. The matrix is preferably in the form of a microparticle such as a microsphere (where the gene is dispersed throughout a solid polymeric matrix) or microcapsule (gene is stored in the core of a polymeric shell), a film, an implant, or a coating on a device such as a stent. The size and composition of the polymeric device is selected to result in favorable release kinetics in tissue. The size is also selected according to the method of delivery which is to be used, typically injection or administration of a suspension by aerosol into the nasal and/or pulmonary areas. The matrix composition can be selected to not only have favorable degradation rates, but to be formed of a material which is bioadhesive, to further increase the effectiveness of transfer when administered to a mucosal surface.

This application is a continuation application under 37 §1.53(b) of U.S.Ser. No. 08/213,668, filed on Mar. 15, 1994 now abandoned, incorporatedherein by reference.

BACKGROUND OF THE INVENTION

The present invention is generally in the area of drug delivery devicesand is specifically in the area of polymeric drug delivery devices.

Gene therapy is generally defined as the introduction and expression ofan exogenous gene in an animal to supplement or replace a defective ormissing gene. Examples that have received a great deal of recentattention include the genes missing in cystic fibrosis and severecombined immunodeficiency. Although tremendous progress has been made inthe area of gene therapy, obtaining long term expression of the desiredproteins remains elusive.

In the majority of cases, a retroviral vector is used to introduce thegene to be expressed into appropriate cells. Gene transfer is mostcommonly achieved through a cell-mediated ex vivo therapy in which cellsfrom the blood or tissue are genetically modified in the laboratory andsubsequently returned to the patient. The clinical studies by StevenRosenberg, et al., “Immunotherapy of patients with metastatic melanomausing tumor-infiltrating lymphocytes and IL-2”, Preliminary report, NewEngland J. Med., 319 (1988) 1676-1680, using in vitro-activated LAK andTIL for tumor destruction illustrates this approach. In other cases, thevector carrying the gene to be expressed is introduced into the patient,for example, by inhalation into the lungs in the case of cysticfibrosis. Transfected cells have also been implanted, alone orencapsulated within a protective membrane that protects the cells fromthe inflammatory response of the body while at the same time allowingthe gene product to diffuse out of the membrane. There have also beenreports of the direct injection of an exogenous gene in combination withan appropriate promoter, into tissue, with some transient expressionbeing noted.

Viral vectors have been widely used in gene transfer, due to therelatively high efficiency of transfection and potential long termeffect through the actual integration into the host's genome. However,there are still concerns about the risks involved in the use of viruses.Activation of proto-oncogenes and reversion to wild-type viruses fromreplication incompetent viruses are some important potential hazards ofviral delivery of genes.

Since the discovery that naked DNA is taken up by muscle cells andtransiently expressed in vivo, and subsequent reports, by Woeff, Jon Aet al., “Direct gene transfer into mouse muscle in vivo,” Science, 247,1465-1468, 1990; and Ascadi et al., “Human dystrophin expression in mdxmice after intramuscular injection of DNA constructs,” Nature, 352,815-818, 1991, there has been increasing interest in using non-viralvehicles for in vivo transfections.

Plasmid DNA, which can function episomally, has been used with liposomeencapsulation, CaPO₄ precipitation and electroporation as an alternativeto viral transfections. Recent clinical trials with liposomeencapsulated DNA in treating melanoma illustrates this approach to genetherapy, as reported by Nabel, J. G., et al., “Direct gene transfer withDNA-liposome complexes in melanoma: Expression, biological activity andlack of toxicity in humans”, Proc. Nat. Acad. Sci. U.S.A., 90 (1993)11307-11311. A foreign gene coding for HLA-B was introduced intosubcutaneous sites of melanoma tumors. Expression of the new gene andthe absence of an anti-DNA host response was confirmed. Wolff, Jon A,“Persistence of plasmid DNA and expression in rat brain cells in vivo,”Experimental Neurology, 115, 400-413, 1992, also reported expression ofplasmid DNA. Thus, direct gene transfer offers the potential tointroduce DNA encoding proteins to treat human diseases.

The mechanisms for cellular uptake of exogenous DNA and subsequentexpression are not clear but gene transfer with naked DNA is associatedwith several characteristics. Unlike in the case of oligonucleotides,which are typically a maximum of twenty to thirty nucleotides in length,genes encoding most molecules of therapeutic interest are quite large,and therefore considerably more difficult to introduce into cells otherthan through retroviral vector, or in vitro, by chemical manipulation,so that the efficiency of transfer is low. In most reported cases todate, only transient expression of up to a few weeks or months has beenobserved. Although low level expression and short term expression aretwo important drawbacks with direct DNA transfer, transfections withnaked DNA have several advantages over viral transfers. Mostimportantly, concerns related to the immunogenicity and transformingcapability of viruses are avoided. In addition, naked DNA is easy toproduce in large quantities, is inexpensive, and can be injected at highconcentration into localized tissue sites allowing gene expression insitu without extensive ex vivo therapy.

The following additional articles review the current state of genetherapy and the problems associated therewith: Blau, Helen M, “Musclingin on gene therapy,” Nature, 364, 673-675, 1993; Cohen, Jon, “Naked DNApoints way to vaccines,” Science, 259, 1691-1692, 1993; Dagani, Ron,“Gene therapy advance, anti-HIV antibodies work inside cells,” C&EN,3-4, 1993; Felgner, Philip L, “Lipofectamine reagent: A new, higherefficiency polycationic liposome transfection reagent,” Focus/Gibco, 15,73-78, 1993; Liu, Margaret A et al, “Heterologous protection againstinfluenza by injection of DNA encoding a viral protein,” Science, 259,1745-1749, 1993; Marx, Jean, “A first step toward gene therapy forhemophilia B,” Science, 262, 29-30, 1993; Mulligan, Richard C, “Thebasic science of gene therapy,” Science, 260, 926-931, 1993; Nicolau,Claude, et al., “In vivo expression of rat insulin after intravenousadministration of the liposome-entrapped gene for rat insulin I,” Proc.Natl. Acad. Sci. USA, 80, 1068-1072, 1983; Partridge, Terence A, “Muscletransfection made easy,” Nature, 352, 757-758, 1991; Wilson, James M,“Vehicles for gene therapy,” Nature, 365, 691-692, 1993; Wivel, et al.,“Germ-line gene modification and disease prevention: Some medical andethical perspectives,” Science, 262, 533-538, 1993; and Woo, Savio L Cet al., “In vivo gene therapy of hemophilia B: sustained partialcorrection in Factor IX-deficient dogs,” Science, 262, 117-119, 1993.

Gene therapy is one of the most promising areas of research today. Itwould therefore be extremely useful if one had an efficient way tointroduce genes into cells which yielded long term expression.

It is therefore an object of the present invention to provide a meansfor efficient transfer of exogenous genes to cells in a patient.

It is a further object of the present invention to provide a means forlong term expression of exogenous genes in patients.

It is a further object of the present invention to provide a means forincreasing or decreasing the inflammatory response to implantedpolymeric devices.

It is a still further object of the present invention to provide amethod for immunization of individuals over a more prolonged period oftime than is achieved by a single or multiple immunization protocol.

It is another object of the present invention to provide a method fortargeting of gene delivery either to tissue cells or to inflammatorytype cells.

SUMMARY OF THE INVENTION

A means for obtaining efficient introduction of exogenous genes into apatient, with long term expression of the gene, is disclosed. The gene,under control of an appropriate promoter for expression in a particularcell type, is encapsulated or dispersed with a biocompatible, preferablybiodegradable polymeric matrix, where the gene is able to diffuse out ofthe matrix over an extended period of time, for example, a period ofthree to twelve months or longer. The matrix is preferably in the formof a microparticle such as a microsphere (where the gene is dispersedthroughout a solid polymeric matrix) or microcapsule (gene is stored inthe core of a polymeric shell), although other forms including films,coatings, gels, implants, and stents can also be used. The size andcomposition of the polymeric device is selected to result in favorablerelease kinetics in tissue. The size is also selected according to themethod of delivery which is to be used, typically injection into atissue or administration of a suspension by aerosol into the nasaland/or pulmonary areas. The matrix composition can be selected to notonly have favorable degradation rates, but to be formed of a materialwhich is bioadhesive, to further increase the effectiveness of transferwhen administered to a mucosal surface, or selected not to degrade butto release by diffusion over an extended period.

Examples demonstrate the effectiveness of the system in animals.

DETAILED DESCRIPTION OF THE INVENTION

Gene transfer is achieved using a polymeric delivery system whichreleases entrapped genes, usually in combination with an appropriatepromoter for expression of the gene, into surrounding tissue. Efficacyof transfer is achieved by: a) releasing the gene for prolonged periodof time, b) minimizing diffusion of the gene out of the delivery system(due to its size) so that release is predominantly degradationdependent, and c) improving the transient time of expression and the lowinfection seen by direct gene therapy. In case of non-erodible polymers,the device is formulated so that the gene is released via diffusion.This is achieved by creating porous systems or adding soluble bulkingagents that create pores as they leach out of the system.

The Polymeric Matrices Selection of Polymer

Both non-biodegradable and biodegradable matrices can be used fordelivery of genes, although biodegradable matrices are preferred. Thesemay be natural or synthetic polymers, although synthetic polymers arepreferred due to the better characterization of degradation and releaseprofiles. The polymer is selected based on the period over which releaseis desired, generally in the range of at least three months to twelvemonths, although longer periods may be desirable. In some cases linearrelease may be most useful, although in others a pulse release or “bulkrelease” may provided more effective results. The polymer may be in theform of a hydrogel (typically in absorbing up to about 90% by weight ofwater), and can optionally be crosslinked with multivalent ions orpolymers.

High molecular weight genes can be delivered partially by diffusion butmainly by degradation of the polymeric system. In this case,biodegradable polymers, bioerodible hydrogels, and protein deliverysystems are particularly preferred. Representative synthetic polymersare: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols,polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols,polyvinyl ethers, polyvinyl esters, polyvinyl halides,polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes andco-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, celluloseethers, cellulose esters, nitro celluloses, polymers of acrylic andmethacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropylcellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methylcellulose, cellulose acetate, cellulose propionate, cellulose acetatebutyrate, cellulose acetate phthalate, carboxylethyl cellulose,cellullose triacetate, cellulose sulphate sodium salt, poly(methylmethacrylate), poly(ethyl methacrylate), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), poly(octadecyl acrylate), polyethylene, polypropylene,poly(ethylene glycol), poly(ethylene oxide), poly(ethyleneterephthalate), poly(vinyl alcohols), poly(vinyl acetate, poly vinylchloride, polystyrene and polyvinylpyrrolidone.

Examples of non-biodegradable polymers include ethylene vinyl acetate,poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.

Examples of biodegradable polymers include synthetic polymers such aspolymers of lactic acid and glycolic acid, polyanhydrides,poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid),and poly (lactide-co-caprolactone), and natural polymers such asalginate and other polysaccharides including dextran and cellulose,collagen, chemical derivatives thereof (substitutions, additions ofchemical groups, for example, alkyl, alkylene, hydroxylations,oxidations, and other modifications routinely made by those skilled inthe art), albumin and other hydrophilic proteins, zein and otherprolamines and hydrophobic proteins, copolymers and mixtures thereof. Ingeneral, these materials degrade either by enzymatic hydrolysis orexposure to water in vivo, by surface or bulk erosion.

Bioadhesive polymers of particular interest include bioerodiblehydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell inMacromolecules, 1993, 26, 581-587, the teachings of which areincorporated herein, polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethyl methacrylates), poly (butylmethacrylate),poly (isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate).

Selection of Matrix Form and Size

In the preferred embodiment, the polymeric matrix is a microparticlebetween nanometers and one millimeter in diameter, more preferablybetween 0.5 and 100 microns for administration via injection orinhalation (aerosol).

The microparticles can be microspheres, where gene is dispersed within asolid polymeric matrix, or microcapsules, where the core is of adifferent material than the polymeric shell, and the gene is dispersedor suspended in the core, which may be liquid or solid in nature. Unlessspecifically defined herein, microparticles, microspheres, andmicrocapsules are used interchangeably.

Alternatively, the polymer may be cast as a thin slab or film, rangingfrom nanometers to four centimeters, a powder produced by grinding orother standard techniques, or even a gel such as a hydrogel. The polymercan also be in the form of a coating or part of a stent or catheter,vascular graft, or other prosthetic device.

Methods for Making the Matrix

The matrices can be formed by solvent evaporation, spray drying, solventextraction and other methods known to those skilled in the art.

Microsphere Preparation

Bioerodible microspheres can be prepared using any of the methodsdeveloped for making microspheres for drug delivery, for example, asdescribed by Mathiowitz and Langer, J. Controlled Release 5,13-22(1987); Mathiowitz, et al., Reactive Polymers 6, 275-283 (1987); andMathiowitz, et al., J. Appl. Polymer Sci. 35, 755-774 (1988), theteachings of which are incorporated herein. The selection of the methoddepends on the polymer selection, the size, external morphology, andcrystallinity that is desired, as described, for example, by Mathiowitz,et al., Scanning Microscopy 4,329-340 (1990); Mathiowitz, et al., J.Appl. Polymer Sci. 45, 125-134 (1992); and Benita, et al., J. Pharm.Sci. 73, 1721-1724 (1984), the teachings of which are incorporatedherein.

In solvent evaporation, described for example, in Mathiowitz, et al.,(1990), Benita, and U.S. Pat. No. 4,272,398 to Jaffe, the polymer isdissolved in a volatile organic solvent. The DNA, either in soluble formor dispersed as fine particles, is added to the polymer solution, andthe mixture is suspended in an aqueous phase that contains a surfaceactive agent such as poly(vinyl alcohol). The resulting emulsion isstirred until most of the organic solvent evaporates, leaving solidmicrospheres.

In general, the polymer can be dissolved in methylene chloride. Severaldifferent polymer concentrations can be used, for example, between 0.05and 0.20 g/ml. After loading the solution with DNA, the solution issuspended in 200 ml of vigorously stirring distilled water containing 1%(w/v) poly(vinyl alcohol) (Sigma Chemical Co., St. Louis, Mo.). Afterfour hours of stirring, the organic solvent will have evaporated fromthe polymer, and the resulting microspheres will be washed with waterand dried overnight in a lyophilizer.

Microspheres with different sizes (1-1000 microns) and morphologies canbe obtained by this method which is useful for relatively stablepolymers such as polyesters and polystyrene. However, labile polymerssuch as polyanhydrides may degrade due to exposure to water. For thesepolymers, hot melt encapsulation and solvent removal may be preferred.

In hot melt encapsulation, the polymer is first melted and then mixedwith the solid particles of DNA, preferably sieved to less than 50 μm.The mixture is suspended in a non-miscible solvent such as silicon oiland, with continuous stirring, heated to 5° C. above the melting pointof the polymer. Once the emulsion is stabilized, it is cooled until thepolymer particles solidify. The resulting microspheres are washed bydecantation with petroleum ether to give a free-flowing powder.Microspheres with diameters between one and 1000 microns can be obtainedwith this method. The external surface of spheres prepared with thistechnique are usually smooth and dense. This procedure is useful withwater labile polymers, but is limited to use with polymers withmolecular weights between 1000 and 50000.

Solvent removal was primarily designed for use with polyanhydrides. Inthis method, the drug is dispersed or dissolved in a solution of aselected polymer in a volatile organic solvent like methylene chloride.The mixture is then suspended in oil, such as silicon oil, by stirring,to form an emulsion. Within 24 hours, the solvent diffuses into the oilphase and the emulsion droplets harden into solid polymer microspheres.Unlike solvent evaporation, this method can be used to make microspheresfrom polymers with high melting points and a wide range of molecularweights. Microspheres having a diameter between one and 300 microns canbe obtained with this procedure. The external morphology of the spheresis highly dependent on the type of polymer used.

In spray drying, the polymer is dissolved in methylene chloride (0.04g/ml). A known amount of active drug is suspended (if insoluble) orco-dissolved (if soluble) in the polymer solution. The solution or thedispersion is then spray-dried. Typical process parameters for amini-spray drier are as follows: polymer concentration=0.04 g/ml, inlettemperature=24° C., outlet temperature=13 to 15° C., aspiratorsetting=15, pump setting=10 ml/min, spray flow=600 NLh⁻¹, and nozzlediameter=0.5 mm. Microspheres ranging in diameter between one and tenmicrons can be obtained with a morphology which depends on the selectionof polymer.

Double walled microspheres can be prepared according to U.S. Pat. No.4,861,627 to Mathiowitz.

Hydrogel microspheres made of gel-type polymers such as alginate orpolyphosphazines or other dicarboxylic polymers can be prepared bydissolving the polymer in an aqueous solution, suspending the materialto be incorporated into the mixture, and extruding the polymer mixturethrough a microdroplet forming device, equipped with a nitrogen gas jet.The resulting microspheres fall into a slowly stirring, ionic hardeningbath, as described, for example, by Salib, et al., PharmazeutischeIndustrie 40-11A, 1230 (1978), the teachings of which are incorporatedherein. The advantage of this system is the ability to further modifythe surface of the microspheres by coating them with polycationicpolymers such as polylysine, after fabrication, for example, asdescribed by Lim, et al., J. Pharm. Sci. 70, 351-354 (1981). Forexample, in the case of alginate, a hydrogel can be formed by tonicallycrosslinking the alginate with calcium ions, then crosslinking the outersurface of the microparticle with a polycation such as polylysine, afterfabrication. The microsphere particle size will be controlled usingvarious size extruders, polymer flow rates and gas flow rates.

Chitosan microspheres can be prepared by dissolving the polymer inacidic solution and crosslinking with tripolyphosphate. For example,carboxymethylcellulose (CMC) microsphere are prepared by dissolving thepolymer in an acid solution and precipitating the microspheres with leadions. Alginate/polyethylene imide (PEI) can be prepared to reduce theamount of carboxyl groups on the alginate microcapsules. Table 1summarizes various hydrogels, concentrations, ionic baths, and stirringrates used to manufacture them.

TABLE 1 Preparation of Hydrogel Matrices Hydrogel dissolving bath ionicbath stirring Hydrogel concen. pH Temp ° C. concen. (w/v) rate chitosan1.0% 5.0 23° C. 3% tripoly- 170 rpm phosphate alginate 2.0% 7.4 50° C.1.3% calcium 160 rpm chloride alginate/ 2.0%/ 7.4 50° C. 1.3% calcium160 rpm PEI 6.0% 7.4 50° C. chloride Carboxy 2.0% 7.4 50° C. 10.0% lead100 rpm methyl nitrate cellulose

Other Device Forms

Other delivery systems including films, coatings, pellets, slabs, anddevices can be fabricated using solvent or melt casting, and extrusion,as well as standard methods for making composites. The polymer can beproduced by first mixing monomers and DNA as described by Sawhney, etal., and polymerizing the monomers with UV light. The polymerization canbe carried out in vitro as well as in vivo. Thus, any biocompatible gluecould be also used to incorporate the DNA.

Loading of Gene

The range of loading of the gene to be delivered is typically betweenabout 0.01% and 90%, depending on the form and size of the gene to bedelivered and the target tissue.

Selection of Genes to be Incorporated

Any genes that would be useful in replacing or supplementing a desiredfunction, or achieving a desired effect such as the inhibition of tumorgrowth, could be introduced using the matrices described herein. As usedherein, a “gene” is an isolated nucleic acid molecule of greater thanthirty nucleotides, preferably one hundred nucleotides or more, inlength.

Examples of genes which replace or supplement function include the genesencoding missing enzymes such as adenosine deaminase (ADA) which hasbeen used in clinical trials to treat ADA deficiency and cofactors suchas insulin and coagulation factor VIII.

Genes which effect regulation can also be administered, alone or incombination with a gene supplementing or replacing a specific function.For example, a gene encoding a protein which suppresses expression of aparticular protein-encoding gene, or vice versa, which induces expressesof a protein-encoding gene, can be administered in the matrix.

Examples of genes which are useful in stimulation of the immune responseinclude viral antigens and tumor antigens, as well as cytokines (tumornecrosis factor) and inducers of cytokines (endotoxin), and variouspharmacological agents.

The chronic immune response to the polymeric matrix is mediated by theaction of a variety of growth factors including epidermal growth factor(EGF), platelet-derived growth factor (PDGF), fibroblast growth factors(FGFs), transforming growth factors (TGF-α and TGF-β, interleukin-1(IL-1), and tumor necrosis factor (TNF). Inhibitors of theseinflammatory mediators in combination with a gene to be delivered otherthan the immune inhibitor would be effective in decreasing the normalinflammatory response directed toward the polymeric matrix. Byinhibiting the amount of encapsulation of the matrix, the effectiverelease would be further extended. Examples of materials which couldinhibit encapsulation include antisense mRNA to suppress fibrin orcollagen formation, inhibitors of EGF, PDGF, FGFs, TGF-α, TGF-β, IL-1and TNF and anti-inflammatory agents such as corticosteroids andcyclosporin.

Genes can be obtained using literature references or from commercialsuppliers. They can be synthesized using solid phase synthesis ifrelatively small, or obtained in expression vectors, for example, asdeposited with the American Type Culture Collection, Rockville, Md.

Selection of Vectors to be Introduced in Combination with the Gene

As used herein, vectors are agents that transport the gene into the cellwithout degradation and include a promoter yielding expression of thegene in the cells into which it is delivered. Promoters can be generalpromoters, yielding expression in a variety of mammalian cells, or cellspecific, or even nuclear versus cytoplasmic specific. These are knownto those skilled in the art and can be constructed using standardmolecular biology protocols. Although as demonstrated by the examples,the genes will diffuse out of the polymeric matrix into the surroundingcells where they are expressed, in a preferred embodiment, the genes aredelivered in combination with a vector to further enhance uptake andexpression. Vectors are divided into two classes:

a) Biological agents derived from viral, bacterial or other sources.

b) Chemical/physical methods that increase the potential for geneuptake, directly introduce the gene into the nucleus or target the geneto a cell receptor.

Biological Vectors

Viral vectors have higher transaction (ability to introduce genes)abilities than do most chemical or physical methods to introduce genesinto cells.

Retroviral vectors are the vectors most commonly used in clinicaltrials, since they carry a larger genetic payload than other viralvectors. However, they are not useful in non-proliferating cells.

Adenovirus vectors are relatively stable and easy to work with, havehigh titers, and can be delivered in aerosol formulation. However, manypeople may have pre-existing antibodies negating effectiveness and theyare difficult to produce in quantity.

Pox viral vectors are large and have several sites for inserting genes,they are thermostable and can be stored at room temperature. However,they cannot be transmitted from host to host and there are some safetyissues since they can enter other cells.

Plasmids are not integrated into the genome and their life span is fromfew weeks to several months, so they are typically very safe. However,they have lower expression levels than retroviruses and since cells havethe ability to identify and eventually shut down foreign geneexpression, the continuous release of DNA from the polymer to the targetcells substantially increases the duration of functional expressionwhile maintaining the benefit of the safety associated with non-viraltransfections.

Chemical/physical Vectors

Other methods to directly introduce genes into cells or exploitreceptors on the surface of cells include the use of liposomes andlipids, ligands for specific cell surface receptors, cell receptors, andcalcium phosphate and other chemical mediators, microinjections directlyto single cells, electroporation and homologous recombination. Thechemical/physical methods have a number of problems, however, and willtypically not be used with the polymeric matrices described herein. Forexample, chemicals mediators are impractical for in vivo use: whencalcium phosphate is used there appears to be very low transductionrate, when sodium butyrate is used the inserted gene is highly unstableand when glycerol is used inserted gene is rapidly lost.

Pharmaceutical Compositions

The microparticles can be suspended in any appropriate pharmaceuticalcarrier, such as saline, for administration to a patient. In the mostpreferred embodiment, the microparticles will be stored in dry orlyophilized form until immediately before administration. They will thenbe suspended in sufficient solution for administration.

In some cases, it may be desirable to administer the microparticles incombination with an adjuvant to enhance the inflammatory responseagainst the polymer and thereby increase the likelihood of phagocytosisby macrophages and other hematopoietic cells, with subsequent expressionof the gene specifically within these cells, or, in the case where themicroparticles contain an anti-cancer agent, to enhance the inflammatoryreaction against the tumor cells in combination with the effect of theanti-cancer agent.

The polymeric microparticles can be administered by injection, infusion,implantation, orally (not preferred), or administration to a mucosalsurface, for example, the nasal-pharyngeal region and/or lungs using anaerosol, or in a cream, ointment, spray, or other topical carrier, forexample, to rectal or vaginal areas. The other devices are preferablyadministered by implantation in the area where release is desired.

The materials can also be incorporated into an appropriate vehicle fortransdermal delivery as well as stents. Appropriate vehicles includeointments, lotions, patches, and other standard delivery means.Targeting of cell populations through polymer material characteristics.

Studies with plasmid release using PLA/PCL biodegradable polymersindicate that the majority of transfected cells, assessed with theβ-galactosidase reporter gene, are inflammatory cells involved in the“foreign body” response. In general, non-degrading polymers evoke astronger inflammatory response when compared to non-biodegradingpolymers. A strong foreign body response results in a thick layer ofmacrophages, fibroblasts, and lymphocytes around the implant. Becausethe polymer release device relies on diffusion for movement of itsparticles, a strong inflammatory response will limit the effectivedistance of diffusion. Accordingly, biodegrading polymers can be used totarget inflammatory cells due to the inability of the plasmid DNA (pDNA)to migrate across the reactive tissue layer to the site specific tissue.A more biocompatible material which induces a weaker response from thehost will result in a thinner layer of inflammatory cells, enabling thereleased pDNA to migrate across the inflammatory cells to the indigenouscells to be transfected.

Incorporation of Antiinflammatories and Immune Enhancers; Treatment ofCancers

In recent years, considerable attention has been focused on the use ofgene therapy to treat various diseases including cancer. Generally, genetherapy for cancer therapeutics either targets the cells of the immunesystem to enhance their ability to kill malignant cells or directlytargets the cancer cells to regulate their proliferation or enhance somecellular function which will result in a stronger activation of theimmune response.

Most types of cancer are characterized by frequent relapses during thecourse of treatment and the continued non-specific and/or specificactivation of the immune system resulting from gene therapy is crucial.Second, cell targeting is a major limitation of current vectors andimplantation of a controlled release device directly inside a tumorwhere the DNA is released locally is one alternative to ex vivo therapyor the development of effective ligand specific vectors. As indicated bythe prevalence of ex vivo therapy, targeting hematopoietic cells isespecially difficult. The histological results from the implant site inthe studies described in the examples below, reveal a substantialinflammatory response surrounding the intramuscular implant. The wellknown “foreign body” host response can be used to an advantage as thismigration of lymphocytes and antigen presenting cells raises thepossibility of directing the transfection to these specific cellpopulations.

Tumors elicit both the humoral and cell-mediated immune response, andlymphocytes, particularly cytotoxic T cells and NK cells, as well asmacrophages, are known to play a crucial role in tumor elimination. Genetherapy for cancer treatment either targets these cells or the malignantcells themselves. An implant releasing naked DNA for long termfunctional gene transfer which can target inflammatory cells and/ortumor cells could significantly improve cancer therapy.

The approaches used include upregulation of class I MHC expression,transduction of antigen presenting cells with tumor-specific antigens,cytokine immunotherapy, transfection of tumor cells with tumorsuppressor genes and anti-sense therapy.

The malignant transformation of cells is often characterized by areduction of class I MHC expression leading to a severe depression ofthe CTL-mediated immune response. An increase in class I MHC expressionon tumor cells could facilitate the activation of the immune systemagainst these altered self-cells. Transfection of genes for cytokinessuch as tumor necrosis factor (TNF) into tumor cells or tumor suppressorgenes such as p53 can be used to limit the ability of tumor cells tomultiply. Anti-sense therapy targets cell proliferation or theproduction of necessary proteins such as tumor angiogenesis factor (TAF)by complementary RNA hybridization to block transcription of specificgenes.

The immune system can be activated and induced to attack specific cellsusing cytokines such as Proleukin or monoclonal antibodies. For example,cancer cells proliferate in part due to a decreased immune responseagainst the transformed cells. The matrices described herein provide ameans to allow recognition and provocation of a response to cancercells. For example, genes coding for antigens; such as the aberrantepithelial mucin of breast cancer, and monoclonal antibodies directedagainst tumor antigens have been shown to have potential in stimulatingimmune destruction of malignant cells. These genes, alone or incombination with monoclonal antibodies, can be delivered to the tumorsites in the polymeric matrices to achieve inhibition of the tumorcells.

Cancer cells can also be treated by introducing chemotherapy drugresistant genes into healthy cells to protect them against the toxicityof drug therapy, or by the insertion of appropriate vectors containingcytotoxic genes or blocking genes into a tumor mass to eliminate cancercells. In a preferred embodiment, the immune system is specificallystimulated against antigens or proteins on the surface of the cancercells.

These approaches can be used in vitro and in vivo. In vitro, the cellscan be removed from a patient, the gene inserted into the cell and thecells reintroduced into the patient. In vivo, the gene can be directlyintroduced into the body either systematically or in localized sites.

Another approach is to use suicide genes that cause cell death when theyare activated or when their product is combined with a pharmaceutical.The primary limitation of the method is the fact that the gene should betargeted to the cancer cell and not to normal cell. Current approach toovercome the problem is direct injection of the vectors into a localizedarea where normal cells do not proliferate. This would be greatlyfacilitated using the polymeric devices described herein. The advantagesof polymeric devices in this setting include continuous and protractedrelease of the incorporated pharmaceutical. This increases the liklihoodthat the intended purposes, for example, treatment of cancerous cells,will be achieved.

EXAMPLES

The method and materials of the present invention will be furtherunderstood by reference to the following non-limiting examples.

Example 1 Expression of Linear and Supercoiled Plasmid DNA Encapsulatedin Polymeric Implants in Muscle Tissue of Rats

The study described in this example confirms the feasibility of in vivotransfections using biodegradable polyester blends to release linear orsupercoiled plasmid DNA. Although only short term expression was studiedin this study, polymer devices releasing drugs offer the potential forsustained long term delivery of naked DNA.

Marker genes are used to study the movement of engineered cellscontaining exogenous genes, as well as the vectors and genes introducedwith the vectors, to insure that the genes remain where they areintroduced. Almost all of the initial research into gene therapy is withmarker genes. Preferred marker genes are those whose product isinnocuous and which can be readily detected by simple laboratory tools.An appropriate marker gene is β-galactosidase (β-gal), since expressionis readily detected by addition of X-gal, a substrate which yields ablue color when the active enzyme is present.

Encapsulation of Linear and Supercoiled β-gal Coding DNA in a PLA Blend

1 g polyactic acid (PLA) (300K) and 2 g PLA (2K) was dissolved in 10 mlof methylene chloride and 5 drops of (SPAN ™) 85. The mixture wasdivided into two aliquots of 5 ml and 100 ul of either circular orlinear DNA (between 1 and 2 mg/ml diluted 1:5 in buffer) was introducedinto the aliquots. Each mixture was mixed well and aliquoted into glassvials (1 ml/vial). Between 20 μg and 40 μg of β-gal plasmid DNA wasencapsulated in each glass vial. The glass vials were left in therefrigerator for four days to evaporate the methylene chloride and thenlyophilized.

Implantation of DNA/PLA pellets

Each sample was first sterilized with ethanol for 5 min and then washedwith PBS-penicillin/streptomycin for 5 min. Surgery was done on SpragueDawley rats. Linear DNA was implanted into the left leg and supercoiledDNA implanted into the right. Implants were inserted into incisedmuscle—either in the vastus or the hamstring. The muscle was suturedback together and then the skin was sutured closed. Rats were sacrificedfor analysis at two weeks.

Results

Rats were perfused with Phosphate Buffered Saline (PBS) with 2500 unitsof heparin followed by 3% paraformaldehyde and 0.2% glutaraldehyde inPBS. The tissue was post-fixed with 3% paraformaldehyde followed by 15%sucrose/PBS. Excised muscles were cut with a cryostat and stained withX-gal.

Histology of the implant sites revealed a substantial inflammatoryresponse around the film at two weeks and two months. The bulk of theβ-gal positive staining was localized to this area with few muscle cellsexhibiting positive staining. The cells present around the implantprobably consists of phagocytic cells, lymphocytes and fibroblasts. Asexpected, transfection was more efficient with supercoiled DNA.

Example 2 In vitro transfection with pRSV β-gal

NIH3T3 fibroblasts were plated onto a 6 well tissue culture dish with 1ml of D-MEM (10% Fetal calf serum with penicillin/streptomycin). 24hours after plating, the cells were transfected with pRSV β-gal controlplasmids as per Promega Profection Mammalian

Transfection system. Plate 1: 10 μl pRSV-Z (3.4 μg) Calcium PhosphatePrecipitated Plate 2: 30 μl pRSV-Z (10.2 μg) Calcium PhosphatePrecipitated Plate 3: 10 μl pRSV-Z (3.4 μg) Naked DNA Plate 4: 30 μlpRSV-Z (10.2 μg) Naked DNA Plate 5: DNA/PLA Plate 6: Control

Plate 5 with the PLA pellet was placed into the well with 4 ml of mediato counter the effect of the decrease in pH. After 24 hours, the DNA/PLApellet was removed and the media left unchanged. At 48 hours, the cellswere fixed and stained with X-Gal (1 ml/plate) overnight.

Results

The efficiency of transfection was very low. All plates except thecontrol well had a handfull of blue staining cells. There was noobservable differences in the number of blue cells among the 5 plates.It was interesting to note that the plate with the DNA/PLA had similarlevels of staining as the other plates even after the fact that half thecells had died and detached due to the PLA degradation.

Example 3 Duration of Expression with pSV β-gal DNA Encapsulated IntoPLA Blends In vitro release of plasmid DNA

pSV β-gal was amplified in HB101 and purified with Qiagen's MEGA PREP™500 μl of plasmid in Tris-EDTA buffer (67.5 μg) was lyophilized andresuspended into 100 μl of sterile dH₂O and incorporated into PLA. 0.05g PLA (2K) and 0.05 g (300K) was dissolved in 1 ml of methylene chlorideand 1 drop of SPAN™ 85. After the polymer was in solution, 100 μl ofplasmid (67.5 μg) was added to the mixture and vortexed for 15 sec. Theresulting film was left in a refrigerator overnight and subsequentlylyophilized overnight.

This film was incubated with 1.0 ml of TE buffer at 37° C. under gentleagitation and sample supernatants tested at 24 hours and at 4 days forthe presence of released DNA. DNA was assayed by agarose gelelectrophoresis on the supernatants.

The results based on the gel of the supernatant after 24 hours ofincubation show that a substantial amount of plasmid was released. After4 days, the results indicate that there was a first phase of release dueto the diffusion of plasmid molecules which are close to the surface ofthe device followed by a slower release at 4 days due to the lowdegradation rate of the polymer which was too low to be measured.

In vivo transfection levels

3 mg PLA (2K) and 1 mg PLA (100K) were dissolved in methylene chloride(0.25 ml). 1 drop of Span™ 85 and 20 μl of plasmid (20 μg) was added tothe solution and homogenized for 1 minute. This solution was air driedin a glass vial for 3 hours in a sterile hood. The brittle film wasground into fine granules and pressed into a pellet form. Three of theseDNA containing pellets were made as well as three control pelletswithout DNA. All pellets were lyophilized overnight to extract residualsolvents.

Three rats received DNA/PLA in their left hamstring and control/PLA intheir right hamstring. Pellets were inserted into incised hamstrings andthe muscles closed with 6-0 Vicryl. Three rats received an injection ofpSV β-gal plasmids (20 μg in 100 μl of TE buffer) over a minute longperiod in their left leg and 100 μl of plain TE buffer in their rightleg as controls. The site of injection was marked with suture.

Rat ID Left Right Implant Duration R112 DNA/PLA Control/DNA 1 week R110DNA/PLA Control/DNA 5 weeks R111 DNA/PLA Control/DNA 10 weeks R115DNA/buffer Control/buffer 1 week R114 DNA/buffer Control/buffer 5 weeks

Rats were perfused with PBS/heparin, followed by 4% paraformaldehyde,and post-fixed in 4% paraformaldehyde followed by 15% and 25%sucrose/PBS. Excised muscles were cut with a cryostat and stained withX-Gal.

Results

In vitro release studies indicate that plasmid DNA can be incorporatedinto polymers without degradation through manufacturing processes andreleased in functional form for possible uptake by surrounding cells.

In vivo studies reveal that with a 20 μg loading of DNA into thepolymer, there is substantial transfection of inflammatory cells at 1and 5 weeks as confirmed by X-gal staining and immunoblotting. At 10weeks, there was no difference in staining intensity between the controlPLA and DNA/PLA. This is believed to be due to the result of the lowloading (20 μg) of the polymer such that after one week the release ratewas below half maximal levels. Investigators using direct injection usedoses in the 100 μg range to see their effects. A higher initialloading, which will lead to continued release of higher amounts of DNAfrom polymers, should prolong transfection durations. Rats injected with20 μg of DNA in solution showed no transfection at 1 and 5 weeks.

Example 4 Comparison of Plasmid DNA Release From Biodegradable andNon-degrading Polymers

Release of plasmids from biodegradable and non-degradable polymer wascompared to test the feasibility of targeting either inflammatory cellsor tissue specific cells by selection of polymer material. Plasmid DNAwas incorporated into a non-degradable elastomer, ethylene vinyl acetatecopolymer (EVAc) and implanted into the same site in different animalsas PLA/PCL implants. EVAc is a very biocompatible polymer which can bemanufactured into a microporous structure through which DNA can diffuseinto the surrounding tissue.

Encapsulation of pRSV β-gal into Polymers

pRSV β-gal in HB101 was purchased from the ATCC (American Type CultureCollection, Rockville, Md.). The plasmids were grown and purified withPromega's Maxi Prep. 1 ml of a 0.1% solution of ELVAX40 (Dupont) inmethylene chloride was vortexed with 645.2 μl of pRSV β-gal (200 μg),frozen in liquid nitrogen and lyophilized. The resulting mixture wasextruded at 55° C. into a rod shaped form.

PLA (2K) and polycaprolactone (PLC) (112K) were dissolved in methylenechloride in a 3:1 ratio and 80 mg of the polymer vortexed with 322.6 μlof pRSV β-gal (100 μg). The mixture was left in the refrigerator for 2days and lyophilized.

Implantation of the Polymers

The EVAc/DNA and PLA/DNA were implanted into rat hamstrings along withtheir control on opposite sides and sacrificed at 2 weeks.

Results

Histological staining with X-gal reveals positive staining of musclecells as well as inflammatory cells in close proximity to the EVAcpolymeric implant at two weeks post-implantation. In comparison, thePLA/PCL implant reveals positive staining of mostly inflammatory cellsonly, in accordance with the earlier data regarding biodegradablepolymers.

Thus the selection of a biodegradable or non-degradable polymer implantcan be used to target delivery to inflammatory cells or tissue cells(for example, muscle). Comparison of PLA/PCL and the EVAc implantsillustrates the different transfected cell populations. Specifically,the PLA/PCL implant results in almost exclusive transfection ofinflammatory cells while the EVAc implant results in a large number oftransfected muscle cells.

Modifications and variations of the method and compositions of thepresent invention will be obvious to those skilled in the art from theforegoing detailed description. Such modifications and variations areintended to come within the scope of the following claims.

We claim:
 1. A preparation of microparticles, each of which preparationof microparticles comprise a synthetic polymeric matrix and an effectiveamount of naked DNA dispersed within the preparation of microparticles,the polymeric matrix being dissolvable in a volatile organic solvent,wherein (a) the size of the microparticles is between 1 and 300 μm indiameter; and (b) the effective amount of naked DNA dispersed within thepreparation of microparticles is greater than 20 μm and wherein the DNAcontains a gene operably linked to a promoter, the nucleotide sequenceof said gene being greater than 30 nucleotides in length, said naked DNAcomprising circular nucleic acid molecules, supercoiled nucleic acidmolecules, or a combination of both.
 2. The preparation ofmicroparticles according to claim 1 wherein the size of themicroparticles is between 1-100 μm.
 3. The preparation of microparticlesaccording to claim 1 wherein the size of the microparticles is between1-10 μm.
 4. The preparation of microparticles according to any one ofclaims 1-5 wherein the polymeric matrix is biodegradable.
 5. Thepreparation of microparticles according to any one of claims 1-5 whereinthe polymeric matrix is non-biodegradable.
 6. The preparation ofmicroparticles according to claim 1 wherein approximately 0.1 -90% byweight of the naked DNA is loaded into the polymeric matrix.
 7. Thepreparation of microparticles of claim 1, wherein the preparation ofmicroparticles contains sufficient entrapped naked DNA such that thenaked DNA can be released for three months following implantation.
 8. Apreparation of microparticles, each of which preparation ofmicroparticles comprises a synthetic polymeric matrix and an effectiveamount of naked DNA dispersed within the preparation of microparticles,the polymeric matrix being dissolvable in a volatile organic solvent,wherein (a) the size of the microparticles is between 1 and 300 μm indiameter; (b) the effective amount of naked DNA dispersed within thepreparation of microparticles is greater than 20 μg and wherein the DNAcontains a gene operably linked to a promoter, the nucleotide sequenceof said gene being greater than 30 nucleotides in length, said naked DNAcomprising circular nucleic acid molecules, supercoiled nucleic acidmolecules, or a combination of both; and (c) the preparation ofmicroparticles contains sufficient entrapped naked DNA such that thenaked DNA can be released for at least 10 weeks following implantation.